Novel analogs of choline for neuroprotection and cognitive enhancement in neurodegenerative disorders

ABSTRACT

The present invention relates to novel analogs of choline and methods of use or treatment of neurodegenerative disorders and/or conditions such as Parkinson&#39;s disease, Huntington disease, Alzheimer&#39;s disease and related disorders such as amyotrophic lateral sclerosis, spinal muscular atrophy, Friedrich&#39;s ataxia, Pick&#39;s disease, Bassen-Kornzweig syndrome, Refsom&#39;s disease, retinal degeneration, Cruetzfelt-Jacob syndrome or prion disease (mad cow disease), dementia with Lewy bodies, schizophrenia, paraneoplastic cerebellar degeneration and neurodegenerative conditions caused by stroke. The present compounds are effective to treat any neurological condition where acetylcholine transmission neurons and their target cells are affected. Compounds according to the present invention are effective to alleviate and/or reverse the effects of a neurodegenerative condition, prevent further deterioration and/or enhance cognition and memory in patients suffering from neurodegenerative disorders, especially Alzheimer&#39;s disease.

RELATED APPLICATIONS

This application claims priority from provisional application Ser. No.60/306,585, filed Jul. 19, 2001.

FIELD OF THE INVENTION

The present invention relates to novel analogs of choline and methods ofuse or treatment of neurodegenerative disorders such as Parkinson'sdisease, Huntington disease, Alzheimer's disease and related disorderssuch as amyotrophic lateral sclerosis, spinal muscular atrophy,Friedrich's ataxia, Pick's disease, Bassen-Kornzweig syndrome, Refsom'sdisease, retinal degeneration, Cruetzfelt-Jacob syndrome or priondisease (mad cow disease), dementia with Lewy bodies, schizophrenia,paraneoplastic cerebellar degeneration and neurodegenerative conditionscaused by stroke. The present compounds are effective to treat anyneurological condition where acetylcholine transmission neurons andtheir target cells are affected. Compounds according to the presentinvention are effective to alleviate and/or reverse the effects of aneurodegenerative condition, prevent further deterioration and/orenhance cognition and memory in patients suffering fromneurodegenerative disorders, especially Alzheimer's disease.

BACKGROUND OF THE INVENTION

As the population ages, the frequency with which patients are diagnosedwith neurodegenerative diseases, especially those which affect mentalfaculties such as Alzheimer's, is growing dramatically. The number ofindividuals having Alzheimer's disease is growing exponentially and itis estimated that today there may be as many as 24 million individualsworldwide afflicted with this condition.

Alzheimer's Disease (AD) is caused by a degenerative process in thepatient which is characterized by progressive loss of cells from thebasal forebrain, cerebral cortex and other brain areas. Acetylcholinetransmitting neurons and their target nerves are particularly affected.Senile plaques and neurofibrillary tangles are present. Pick's diseasehas a similar clinical picture to Alzheimer's disease but a somewhatslower clinical course and circumscribed atrophy, mainly affecting thefrontal and temporal lobes. One animal model for Alzheimer's disease andother dementias displays hereditary tendency toward the formation ofsuch plaques. It is thought that if a drug has an effect in the model,it also may be beneficial in at least some forms of Alzheimer's andPick's diseases. At present there are palliative treatments but no meansto restore function in Alzheimer's patients.

Parkinson's disease (PD), is a disorder of middle or late life, withvery gradual progression and a prolonged course. HARRISON'S PRINCIPLESOF INTERNAL MEDICINE, Vol. 2, 23d ed., Ed by Isselbacher, Braunwald,Wilson, Martin, Fauci and Kasper, McGraw-Hill Inc., New York City, 1994,pg. 2275. The most regularly observed changes in patients withParkinson's disease have been in the aggregates of melanin-containingnerve cells in the brainstem (substantia nigra, locus 20 coeruleus),where there are varying degrees of nerve cell loss with reactive gliosis(most pronounced in the substantia nigra) along with distinctiveeosinophilic intracytoplasmic inclusions. (Id. at 2276). In its fullydeveloped form, PD is easily recognized in patients, where stoopedposture, stiffness and slowness of movement, fixity of facialexpression, rhythmic tremor of the limbs, which subsides on activewilled movement or complete relaxation, are common features. Generally,accompanying the other characteristics of the fully developed disorderis the festinating gait, whereby the patient, progresses or walks withquick shuffling steps at an accelerating pace as if to catch up with thebody's center of gravity. (Id. At 2276).

The treatment of Parkinson's disease pharmacologically with levodopacombined with stereotactic surgery has only represented a partial cure,at best. (Id at 2277). Underlying much of the treatment difficulty isdirected to the fact that none of these therapeutic measures has aneffect on the underlying disease process, which consists of neuronaldegeneration. Ultimately, a point seems to be reached where pharmacologycan no longer compensate for the loss of basal ganglia dopamine. (Id.).

A group of related neuronal degenerative disorders is characterized byprogressive ataxia due to degeneration of the cerebellum, brainstem,spinal cord and peripheral nerves, and occasionally the basal ganglia.Many of these syndromes are hereditary; others occur sporadically. Thespinocerebellar degenerations are logically placed in three groups:predominantly spinal ataxias, cerebellar ataxias and multiple-systemdegenerations. To date there are no treatments. Friedrich's ataxia isthe prototypical spinal ataxia whose inheritance is autosomal recessive.The responsible gene has been found on Chromosome 9. Symptoms beginbetween ages of 5 and 15 with unsteady gait, followed by upper extremityataxia and dysarthria. Patients are flexic and lose large-fiber sensorymodalities (vibration and position sense). Two other diseases havesimilar symptoms: Bassen-Kornzweig syndrome (abeta-lipoproteinemia andvitamin E deficiency) and Refsom's disease (phytanic acid storagedisease). Cerebellar cortical degenerations generally occur between ages30 and 50. Clinically only signs of cerebellar dysfunction can bedetected, with pathologic changes restricted to the cerebellum andoccasionally the inferior olives. Inherited and sporadic cases have beenreported. Similar degeneration may also be associated with chronicalcoholism. In multiple-system degenerations, ataxia occurs in young tomiddle adult life in varying combinations with spasticity andextrapyramidal, sensory, lower motor neuron and autonomic dysfunction.In some families, there may also be optic atrophy, retinitis pigmentosa,opthalmoplegia and dementia.

Another form of cerebellar degeneration is paraneoplastic cerebellardegeneration that occurs with certain cancers, such as oat cell lungcancer, breast cancer and ovarian cancer. In some cases, the ataxia mayprecede the discovery of the cancer by weeks to years. Purkinje cellsare permanently lost, resulting in ataxia. Even if the patient ispermanently cured of the cancer, their ability to function may beprofoundly disabled by the loss of Purkinje cells. There is no specifictreatment. Stroke often also results in neuronal degeneration and lossof functional synapses.

OBJECT OF THE INVENTION

It is an object of the invention to provide novel choline analogs whichcan be used to treat neurological conditions where acetylcholinetransmission neurons and their target cells are affected.

It is another object of the invention to provide pharmaceuticalcompositions which may be used to treat any one or moreneurodegenerative disease and conditions including, for example,Parkinson's disease, Huntington disease, Alzheimer's disease and relateddisorders such as amyotrophic lateral sclerosis, spinal muscularatrophy, Friedrich's ataxia, Pick's disease, Bassen-Kornzweig syndrome,Refsom's disease, dementia with Lewy bodies, schizophrenia,paraneoplastic cerebellar degeneration and neurodegenerative conditionscaused by stroke.

It is still another object of the invention to provide compositions andmethods which are effective to reverse the effects of aneurodegenerative condition, prevent further deterioration and/orenhance cognition and memory in patients suffering fromneurodegenerative disorders, especially including Alzheimer's disease.

One or more of these and/or other objects of the present invention maybe readily gleaned from the description of the invention which follows.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1-4 are representative of synthetic chemical schemes which may beused to prepare compounds according to the present invention.

FIG. 5 shows the results of the MTT assay from the experimental sectionunder control conditions, and in cultures in which a choline analog wasintroduced into the medium at the time of neurotrophic factorwithdrawal. The data for choline and 6 other analogs are presented.

FIG. 6 shows the results of data from the MTT reduction assay from theexperimental section. As indicated, co-administration of choline and thenon-selective antagonist mecamylamine resulted in a significant (andalmost complete) inhibition of choline's neuroprotective action. Asimilar finding was obtained for those experiments in which theantagonist, MLA, was used.

FIG. 7 shows an experiment which measured the ability of choline todisplace the cell surface binding of [¹²⁵I]-bungarotoxin (a selective 7nicotinic acetylcholine receptor antagonist) to differentiated PC-12cells. These data are presented along with the displacement curve fornicotine as a comparison.

FIG. 8 shows an experiment which measured the ability ofpyrrolidinecholine, an active choline analog, compared withbenzylcholine, the inactive choline analog in the neuroprotection assay,for their ability to displace the cell surface binding of[¹²⁵I]-bungarotoxin to PC-12 cells.

FIGS. 9 and 10, show that the neuroprotective action ofpyrrolidinecholine (like choline) (FIG. 9) was blocked by pretreatmentwith either mecamylamine (10 M) or MLA (10 nM) (FIG. 10).

FIG. 11 shows the cytoprotective effect of nicotine in differentiatedPC-12 cells. The % Protection values presented were calculated as theratio of ELISA-based absorbance values for [protected cells-deprivedcells (no nicotine): control (non-deprived) cells—deprived cells]×100.

FIG. 12 shows a number of compounds according to the present inventionas pharmaceutically acceptable salts.

FIGS. 13A and B show the cytoprotective effect of nicotine analogs indifferentiated PC-12 cells.

SUMMARY OF THE INVENTION

The present invention relates to compounds according to the structures:

Where each of R¹, R² and R⁴ is independently selected from H, a C₁ toC₁₂ straight, branch-chained or cyclic saturated or unsaturatedhydrocarbon, a (CH₂)_(n)OR⁵ group or an R⁶ group; R³ is H, a C₁ to C₁₂alkyl or alkene group or a (CH₂)_(n)OR⁵ group with the proviso that R³and R⁴ are not both H;

-   R^(A) and R^(B) are independently selected from H, a C₁ to C₁₂ alkyl    or alkenyl group or a (CH₂)_(m)OR⁵ group, preferably with the    proviso that when one of R^(A) or R^(B) is a (CH₂)_(m)OR⁵ group, the    other of R^(A) or R^(B) is not a (CH₂)_(m)OR⁵ group;    R⁵ is H or a C₁ to C₁₂ alkyl, alkenyl group or carboxylic acid, a C₂    to C₁₂ acyl or alkyl ester group or a C₃ to C₁₂ alkylene ester    group;    R⁶ is a group according to the structure:

Z is

n is 0 to 12, preferably 1 to 8;m is 1 to 8; and

-   X₁, X₂ and X₃ are each independently selected from H, OH, F, Cl, Br,    NO₂, R⁴, OR⁴, CF₃ or OCF₃;    or a pharmaceutically acceptable salt thereof.

Compounds according to the present invention may be used to producepharmaceutical compositions for treatment of one or moreneurodegenerative diseases or conditions, for example, Parkinson'sdisease, Huntington disease, Alzheimer's disease and other disorderssuch as amyotrophic lateral sclerosis, spinal muscular atrophy,Friedrich's ataxia, Pick's disease, Bassen-Kornzweig syndrome, Refsom'sdisease, retinal degeneration, Cruetzfelt-Jacob syndrome or priondisease (mad cow disease), dementia with Lewy bodies, schizophrenia,paraneoplastic cerebellar degeneration and neurodegenerative conditionscaused by stroke. The present compounds are effective to treat anyneurological condition where acetylcholine transmission neurons andtheir target cells are affected. Compounds according to the presentinvention are effective to reverse the effects of a neurodegenerativecondition, prevent further deterioration and/or enhance cognition andmemory, especially in patients suffering from neurodegenerativedisorders, such as Alzheimer's disease.

Compounds according to the present invention may also be used asbiological probes or standards for testing purposes or as intermediatesin the synthesis of related compounds having pharmacological activity.

The present invention is also directed to pharmaceutical compositionscomprising effective amounts of any one or of the compounds according tothe present invention or their pharmaceutically acceptable derivatives,including pharmaceutically acceptable salts, optionally, in combinationwith a pharmaceutically acceptable additive, carrier or excipient.

The present invention is also directed to methods for the treatment ofneurological conditions where acetylcholine transmission neurons andtheir target cells are affected. Thus, the present compounds mayfunction primarily to reverse and/or prevent deterioration from aneurodegenerative condition. The present compounds may also be used toenhance cognition in memory in patients whose cognition and/or memory isimpaired, either due to natural aging processes or to aneurodegenerative condtion. In this aspect according to the presentinvention, the use of pyrrolidine choline or pyrrolidine acetyl cholineis preferred.

DETAILED DESCRIPTION OF THE INVENTION

The term “patient” is used throughout the specification to describe ananimal, preferably a human, to whom treatment, including prophylactictreatment, with the compositions according to the present invention, isprovided. For treatment of those infections, conditions or diseasestates which are specific for a specific animal such as a human patient,the term patient refers to that specific animal.

The term “neurodegenerative disease” is used throughout thespecification to describe a disease or condition of the nervous systemin which the nervous system often deteriorates over time, thus impairingthe patient from carrying out normal tasks including motor tasks andtasks related to cognition and/or memory. Neurodegenerative diseaseswhich may be treated using compounds according to the present inventioninclude, for example, Parkinson's disease, Huntington disease,Alzheimer's disease and related disorders such as amyotrophic lateralsclerosis, spinal muscular atrophy, Friedrich's ataxia, Pick's disease,Bassen-Kornzweig syndrome, Refsom's disease, retinal degeneration,Cruetzfelt-Jacob syndrome or prion disease (mad cow disease), dementiawith Lewy bodies, schizophrenia, paraneoplastic cerebellar degenerationand neurodegenerative conditions caused by stroke, among others. Aneurodegenerative disease for purposes of the present invention is anyneurological condition where acetylcholine transmission neurons andtheir target cells are affected, including neurological conditions whichare caused by toxins.

The term “treatment”, “treating” or “treated” is used throughout thespecification to describe a method in which compounds according to thepresent invention are used to reverse the effects of a neurodegenerativecondition, prevent further deterioration and/or enhance cognition andmemory, especially in patients suffering from neurodegenerative orneurologicial disorders, such as Alzheimer's disease or those which havebeen caused by toxins or occurred from natural causes such as aging.

The term “effective amount” is used throughout the specification to meanan amount or concentration of a compound according to the presentinvention which is effective within the context of its administration,whether that context produces the desired result of alleviating,reversing or preventing further deterioration of the condition ordisease state to be treated. Effective amounts of compounds according tothe present invention include those amounts which are effective toenhance and/or increase cognition and/or memory in patients in need.

The term “cognitive task” or “cognitive function” is used to describe anendeavor or process by a patient or subject which involves thought orknowing by which animals, particularly humans, come to know the world.Selectively attending to a particular stimulus, recognizing andidentifying these relevant stimulus features and planning andexperiencing the response are some of the processes or abilitiesmediated by the human brain which are related to cognition.

The term “memory” is used to describe the faculty of remembering—thepower with which an individual reproduces past impressions, e.g., athought process or visual or sensory information to which a patient hasbeen exposed in the past and which the patient is able to recall,assimilate and use for a purpose in the present.

The term “motor task” is used to describe an endeavor which is taken bya patient or subject which involves movement or action.

The term “perceptual task” is used to describe an act by a patient orsubject of devoting attention to sensory inputs.

The term “impaired” where used, describes a function of the neurologicalsystem which is working at a level which is less than normal. Impairedfunctions can be significantly impacted such that a function is barelybeing carried out, is virtually non-existent or is working in a fashionwhich is significantly less than normal. The impairment of function willvary in type as well as severity from patient to patient and thecondition to be treated.

The term “pharmaceutically acceptable derivative” is used throughout thespecification to describe any pharmaceutically acceptable salt orprodrug form which, upon administration to a patient, provides directlyor indirectly the compound or an active metabolite of the compoundaccording to the present invention. In general, the free amine form(generally, a secondary or tertiary amine) of compounds according to thepresent invention readily form salts with organic and/or mineral acids.Pharmaceutically acceptable salts include those derived frompharmaceutically acceptable inorganic or organic bases and acids,generally, in the case of the present invention, inorganic and organicacids (due to the presence of an amine group which may be protonated).Suitable salts preferably include ammonium salts obtained fromacidifying amine groups of the compound with organic and inorganic acidswell known in the pharmaceutical art.

The term “alkyl” shall mean within its context a fully saturatedhydrocarbon which may be linear, branch-chained or cyclic radical withinits context, preferably a C₁-C₄, even more preferably a C₁-C₃ linear,branch-chained or cyclic fully saturated hydrocarbon radical. The term“alkenyl” is used to describe a hydrocarbon group, similar to an alkylgroup which contains one double bond.

The term “acyl” is used to describe a group having a carbonyl group towhich is bonded carbon atoms. The term “ester” is used to describe analkoxy carbonyl group. The term carboxylic acid is used to describe acarboxylic acid or alkylene carboxylic acid group. The terms acyl, esterand carboxylic acid includes alkyl acyl groups, alkyl ester groups,alkylene alkyl acyl groups or alkylene alkyl ester groups of theformula:

Where n is 0 to 11 and R is H or a C₁-C₁₁ alkyl group.

Compounds according to the present invention are used to treatneurodegenerative diseases and conditions by alleviating, reversing orpreventing further deterioration from diseases or conditions such asParkinson's disease, Huntington disease, Alzheimer's disease and relateddisorders such as amyotrophic lateral sclerosis, spinal muscularatrophy, Friedrich's ataxia, Pick's disease, Bassen-Kornzweig syndrome,Refsom's disease, retinal degeneration, Cruetzfelt-Jacob syndrome orprion disease (mad cow disease), paraneoplastic cerebellar degenerationand neurodegenerative conditions caused by stroke. The present compoundsmay be used to treat any neurological condition where acetylcholinetransmission neurons and their target cells are affected.

Methods for treating conditions or disease states as described abovecomprise administering an effective amount of at least one or morecompounds according to the present invention to a patient in needthereof to alleviate, reverse and/or prevent or reduce the likelihoodthat further deterioration will occur in the condition or disease statewhich is treated. In the case of treating a patient to improve thatpatient's memory and/or cognition or prevent further deterioration ofmemory or cognition, such methods comprise administering to a patient inneed thereof an effective amount of one or more of the compoundsaccording to the present invention.

General Chemical Synthesis

The compounds according to the present invention are produced bysynthetic methods which are readily known to those of ordinary skill inthe art and include various chemical synthetic methods as elaborated insignificantly more detail in the Examples which follow. In general,compounds according to the present invention are synthesized by methodswhich are well-known in the art.

For example, as set forth in Scheme I, the base catalzyed condensationof ester (1) and N-vinylpyrrolidinone may be accomplished underanhydrous reflux conditions in either toluene, tetrahydrofuran orsimilar aprotic solvent in the presence of strong non-nucleophilic basesuch as sodium hydride, sodium bis(trimethylsilyl)amide, lithiumdi-isopropylamide, lithium bis(trimethylsilyl)amide to provide theketolactam (2). Introduction of an R group at the 3 position of thepyrrolidine ring is accomplished by base generated anion formationfollowed by alkylation with an R—X (X is a leaving group) company togive compound 3. Treatment of compound 3 with aqueous acid under refluxconditions generates compound 4. Reduction of compound 4 with sodiumborohydride in methanol/acetic acid (4:1) at −15 to 0 C yields compound5. Reductive N-alkylation with benzyloxy aldehyde in the presence ofeither sodium cyanoborohydride or sodium triacetoxyborohydride (orequivalent conditions) yields compound 6 which is de-benzylated usingcatalytic hydrogenation to compound 8. Alternatively, compound 5 can beconverted to compound 7 by refluxing in a mixture of formic acid andformaldehyde as indicated in Scheme I.

Compounds which contain unsaturation in the pyrrolidine ring or in theside chain on position 3 may be synthesized readily by an alternativeroute following the steps which are set forth in Scheme II or using anyanalogous procedure known in the art. The ketolactam (2) is converted tothe imine (9) by refluxing in aqueous acid. Reaction of compound (9)with aldehydes under acidic conditions generates compound (10).Reduction of compound (10) with, for example, sodium borohydride, givescompound (11), which undergoes reductive N-alkylation as described abovewith benzyloxy aldehyde to provide compounds (12) and (14). Othercompounds within the scope of the present invention may be readilysynthesized by analogy using the above-described general methods and asset forth in the examples section which follows.

Piperidine, piperazine and other diamine analogs such aspyridylpiperazine compounds of the present invention may be synthesizedaccording to scheme 3 set forth in FIG. 3. Pursuant to scheme 3, thecorresponding formyl pyridine analog is reacted with anamine-substituted piperazine compound under condensation conditions (inscheme 3, using for example, NaBH(AcO)₃ in the presence oftetrahydrofuran at reduced temperature) to afford the correspondingpyridinepiperazine compound. Analogs with numerous substituents oneither the pyridine ring or the piperidine or piperazine (orcorresponding unsaturated amine or diamine) ring

Other compounds which contain tertiary amine groups pendant to an arylgroup, such as benzene are readily synthesized according to syntheticscheme 4 set forth in FIG. 4 or by modifications thereof. See, forexample, A. F. Abdel-Magid, K. G. Carson, B. D. Harris, C. A. Marynoffand R. D. Shah, “Reductive Amination of Aldehydes and Ketones withSodium Triacetoxyborohydride. Studies on Direct and Indirect ReductiveAmination Procedures,” J. Org. Chem., 61, 3849, (1996).

According to the scheme set forth in FIG. 4, the appropriate benzyl orpyridylaldehyde (21-24) is reacted with methylethanolamine to rapidlyform the corresponding imine, which is then subjected to reducingconditions in the presence of NaBH(AcO)₃ in 1,2-dichloroethane or THF,to form the corresponding benzyl substituted tertiary alkanolamine(25-28), which may readily esterified using standard techniques (forexample, acetic anhydride in sodium acetate).

During chemical synthesis of the various compositions according to thepresent invention, one of ordinary skill in the art will be able topractice the present invention without undue experimentation. Inparticular, one of ordinary skill in the art will recognize the varioussteps that should be performed to introduce a particular substituent atthe desired position of the pyrrolidine ring or related synthon.

Pharmaceutical compositions according to the present invention comprisethe above-described compounds in a therapeutically effective amount fortreating a neurodegenerative disease or condition or for enhancing orrestoring or preventing the deterioration of cognition and/or memory ina patient, optionally in combination with a pharmaceutically acceptableadditive, carrier or excipient. One of ordinary skill in the art willrecognize that a therapeutically effective amount will vary with thedisease or condition to be treated, its severity, the treatment regimento be employed, the pharmacokinetics of the agent used, as well as thepatient (animal or human) treated.

In the pharmaceutical aspect according to the present invention, thecompound according to the present invention is formulated preferably inadmixture with a pharmaceutically acceptable carrier. In general, it ispreferable to administer the pharmaceutical composition inorally-administrable form, but certain formulations may be administeredvia a parenteral, intravenous, intramuscular, transdermal, buccal,subcutaneous, suppository or other route. Intravenous and intramuscularformulations are preferably administered in sterile saline. Of course,one of ordinary skill in the art may modify the formulations within theteachings of the specification to provide numerous formulations for aparticular route of administration without rendering the compositions ofthe present invention unstable or compromising their therapeuticactivity. In particular, the modification of the present compounds torender them more soluble in water or other vehicle, for example, may beeasily accomplished by minor modifications (salt formulation,esterification, etc.) which are well within the ordinary skill in theart. It is also well within the routineer's skill to modify the route ofadministration and dosage regimen of a particular compound in order tomanage the pharmacokinetics of the present compounds for maximumbeneficial effect in patients.

In certain pharmaceutical dosage forms, the pro-drug form of thecompounds, especially including ester and ether derivatives, as well asvarious salt forms of the present compounds, are preferred. One ofordinary skill in the art will recognize how to readily modify thepresent compounds to pro-drug forms to facilitate delivery of activecompounds to a targeted site within the host organism or patient. Theroutineer also will take advantage of favorable pharmacokineticparameters of the pro-drug forms, where applicable, in delivering thepresent compounds to a targeted site within the host organism or patientto maximize the intended effect of the compound.

The amount of compound included within therapeutically activeformulations according to the present invention is an effective amountfor treating the infection or condition. In general, an effective amountof the present compound in pharmaceutical dosage form usually rangesfrom about 0.05 mg/kg to about 100 mg/kg or more, more preferably,slightly less than about 0.1 mg./kg. to about 20 mg./kg. of the patientor considerably more, depending upon the compound used, the condition orinfection treated and the route of administration. For purposes of thepresent invention, a prophylactically or preventive effective amount ofthe compositions according to the present invention (i.e., an amountwhich substantially reduces the risk that a patient will either succumbto a disease state or condition or that the disease state or conditionwill worsen) falls within the same concentration range as set forthabove for therapeutically effective amounts and is usually the same as atherapeutically effective amount.

Administration of the active compound may range from continuous(intravenous drip) to several oral administrations per day (for example,Q.I.D.) and may include oral, topical, parenteral, intramuscular,intravenous, sub-cutaneous, transdermal (which may include a penetrationenhancement agent), buccal and suppository administration, among otherroutes of administration. Enteric coated oral tablets may also be usedto enhance bioavailability of the compounds from an oral route ofadministration. The most effective dosage form will depend upon thepharmacokinetics of the particular agent chosen as well as the severityof disease in the patient. Oral dosage forms are particularly preferred,because of ease of admnistration and prospective favorable patientcompliance.

To prepare the pharmaceutical compositions according to the presentinvention, a therapeutically effective amount of one or more of thecompounds according to the present invention is preferably intimatelyadmixed with a pharmaceutically acceptable carrier according toconventional pharmaceutical compounding techniques to produce a dose. Acarrier may take a wide variety of forms depending on the form ofpreparation desired for administration, e.g., oral or parenteral. Inpreparing pharmaceutical compositions in oral dosage form, any of theusual pharmaceutical media may be used. Thus, for liquid oralpreparations such as suspensions, elixirs and solutions, suitablecarriers and additives including water, glycols, oils, alcohols,flavouring agents, preservatives, colouring agents and the like may beused. For solid oral preparations such as powders, tablets, capsules,and for solid preparations such as suppositories, suitable carriers andadditives including starches, sugar carriers, such as dextrose,mannitol, lactose and related carriers, diluents, granulating agents,lubricants, binders, disintegrating agents and the like may be used. Ifdesired, the tablets or capsules may be enteric-coated or sustainedrelease by standard techniques. The use of these dosage forms maysignificantly the bioavailability of the compounds in the patient.

For parenteral formulations, the carrier will usually comprise sterilewater or aqueous sodium chloride solution, though other ingredients,including those which aid dispersion, also may be included. Of course,where sterile water is to be used and maintained as sterile, thecompositions and carriers must also be sterilized. Injectablesuspensions may also be prepared, in which case appropriate liquidcarriers, suspending agents and the like may be employed.

Liposomal suspensions (including liposomes targeted to specific antigenssuch as receptors for more effective delivery to a site within apatient's body) may also be prepared by conventional methods to producepharmaceutically acceptable carriers. This may be appropriate for thedelivery of a number of compounds according to the present invention.

In preferred embodiments according to the present invention, thecompounds and compositions are used to treat, prevent or delay the onsetof Alzheimer's disease or to improve the memory and/or cognition ofpatients suffering from Alzheimer's disease. Preferably, to treat,prevent or delay the onset of Alzheimer's disease or its relatedsymptomotology, the compositions according to the present invention willbe administered in oral dosage form in amounts ranging from about 250micrograms up to about 500 mg or more at least once a day, preferably,up to four times a day. The present compounds are preferablyadministered orally, but may be administered parenterally, topically orin suppository form.

Some of the compounds according to the present invention, because oftheir low toxicity to host cells, may advantageously be employedprophylactically. In this aspect according to the present invention, thepresent compositions are used to prevent or delay the onset of aneurodegenerative disease or prevent the patient from furtherdeterioration from the disease. This prophylactic method comprisesadministering to a patient in need of such treatment or who is at riskfor the development of neurodegenerative disease and in particular,Alzheimer's disease, an amount of a compound according to the presentinvention effective for alleviating, preventing or delaying the onset ofthe disease. In the prophylactic treatment according to the presentinvention, it is preferred that the compound utilized should be as lowin toxicity and preferably non-toxic to the patient. It is particularlypreferred in this aspect of the present invention that the compoundwhich is used should be maximally effective against the disease stateand should exhibit a minimum of toxicity to the patient. In the case ofthe present compound for the prophylactic aspect of the presentinvention, these compounds may be administered within the same dosagerange for therapeutic treatment (i.e., about 250 micrograms up to about500 mg. or more from one to four times per day for an oral dosage form)as a prophylactic agent to prevent or delay the onset of thesymptomotology of the neurodegenerative diseases, and in particular,Alzheimer's disease which manifests itself in clinical symptoms.

In addition, compounds according to the present invention may beadministered alone or in combination with other agents, including othercompounds of the present invention. Certain compounds according to thepresent invention may be effective for enhancing the biological activityof certain agents according to the present invention by reducing themetabolism, catabolism or inactivation of other compounds and as such,are co-administered for this intended effect.

As indicated, compounds according to the present invention may beadministered alone or in combination with other agents having similar orpreferably a different mechanism of action, which may be used fortreating neurodegenerative diseases or related conditions. Certaincompounds according to the present invention may be effective forenhancing the biological activity of certain agents according to thepresent invention by reducing the metabolism or inactivation of othercompounds and as such, are co-administered for this intended effect.

While not being limited by way of theory or mechanism of action, it isbelieved that the compounds according to the present invention exhibittheir pharmacological activity by acting as agonists of theacetylcholine nicotinic receptor in order to enhance cognition and/ormemory or to protect cells against various types of toxic insults.

The present invention is now described, purely by way of illustration,in the following examples. It will be understood by one of ordinaryskill in the art that these examples are in no way limiting and thatvariations of detail can be made without departing from the spirit andscope of the present invention.

EXAMPLES CHEMICAL SYNTHESIS OF COMPOUNDS

General procedure for preparation of ketolactam (Brandage and Lindblom,Acta Chem. Scand., 30, 93, 1976 and Bleicher, et al., J. Org. Chem. 63,1109-1118, 1998) (2) To NaH (1.1 equiv., 1.4 g), rinsed with toluene(3×) to remove mineral oil, stirring in approximately 10 ml toluene wasadded neat N-vinyl pyrrolidinone 99% (1.1 equiv.). After stirring for 10mins., ester dissolved in toluene was added dropwise. Upon addition ofthe ester, an exotherm evolved and the reaction mixture became green incolor. After the exotherm subsided, the reaction stirred for anadditional 30 mins. at ambient temperature and during this time thecolor changed to yellow. The reaction was heated to reflux for 15 mins.,and slowly cooled to room temperature. Water was poured onto thereaction to dissolve the orange, pasty product. The aqueous layer waswashed with diethyl ether, and neutralized with 5% HCl. The neutralaqueous phase was extracted with EtOAc (3×), and the organic extractspooled and concentrated to yield the crude ketolactam product, which wassubsequently used without purification.

3-(1-pyrrolin-2-ylmethyl)pyridine (homomyosmine) (Brandage and Lindblom,Acta Chem. Scand., 30, 93, 1976 and Bleicher, et al., J. Org. Chem. 63,1109-1118, 1998) To a 500 ml round-bottom flask equipped with aDean-Stark apparatus and a non-pressure regulated addition funnel wasadded approximately 200 m150% HCl solution. The acid solution was heatedto 70° C. and the crude ketolactam product dissolved in approximately 10ml THF was added dropwise to the stirring acid solution. After additionof 2, the Dean-Stark trap and the addition funnel were removed and awater condenser was connected to the reaction flask. The reaction washeated to reflux for 18 h. After reflux, the reaction mixture haddarkened and a tarry byproduct had formed on the vessel wall. Thereaction was slowly cooled to room temperature and washed with EtOAc.The aqueous phase was cooled in an ice bath and 50% NaOH solution wascarefully poured onto the solution. As the pH approached neutrality, thesolution became opaque. Base was added until pH˜9, and the aqueous layerwas extracted with EtOAc. The organic layer was washed consecutivelywith H₂O and brine, and dried over sodium sulfate. Filtration andevaporation of the solvent yielded the crude imine product as an ambersyrup. The reaction product was eluted with chloroform through a 1-inchpad of Al (reactivity III) to remove polar contaminants. The eluate wasconcentrated to a dark amber syrup, which was redissolved in EtOAc and asaturated solution of oxalic acid/EtOAc was added dropwise to themixture. After trituration of the product in the acid solution, theoxalate salt precipitated from the solution and the solids collected byvacuum filtration. The pale yellow powder was recyrstallized fromisopropanol/MeOH as an off-white powder (2.8 g, 34%—over two steps).

NMR free base ¹H (CDCl₃): 1.86 (m, 2H, H-9a, 9b), 2.43 (t, J_(8,9)=8,2H, H-8a, 8b), 3.68 (s, 2H, H-6a, 6b), 3.83 (m, 2H, H-10a, 10b), 7.26(dd, J_(3,4; 4,5)=4, 1H, H-4), 7.59 (d, J_(3,4)=4, 1H, H-3), 8.49 (t,2H, H-1, 5). free base ¹³C (CDCl₃): 22.50 (C-6), 36.70 (C-9), 37.54(C-8), 60.92 (C-10), 123.41 (C-4), 132.45 (C-2), 136.48 (C-3), 148.03(C-5), 150.11 (C-1), 175.54 (C-7). (M+H)⁺161.11, m.p. oxalate 123° C.

3-(pyrrolidin-2-ylmethyl)pyridine (norhomonicotine) (5) To the oxalatesalt of 4 (1.5 g, 6.0 mmol) dissolved in MeOH/AcOH (4:1) and stirring at−15° C. (ice/brine) was added excess NaBH₄ over a period of 30 minutes.The reaction continued to stir and was allowed to warm to ambienttemperature overnight. To the reaction was added HCl until pH˜2. Thereaction mixture was washed with Et₂O, and the aqueous phase separatedand basified with NaOH until pH˜13. The aqueous phase was extracted withCHCl₃ and the organic layer separated and dried over sodium sulfate.After filtration, the solvent was evaporated and the residue taken up inEtOAc. To the product solution was added dropwise a solution of HBr inEtOH. The solution was concentrated by rotary evaporation (2×), whichresulted in a powdery residue. The HBr salt of 3 was recrystallized fromEtOH as an off-white powder (1.3 g, 91%).

NMR HBr ¹H (MeOH): 1.86 (m, 1H, H-9a), 2.07 (m, 1H, H-9b), 2.17 (m, 1H,H-8a), 2.26 (m, 1H, H-8b), 3.28-3.57 (m, 4H, H-6a, 6b, 10a, 10b), 4.04(m, 1H, H-7), 8.13 (dd, J_(3,4)=8, J₄₋₅=5.7, 1H, H-4), 8.71 (d,J_(3,4)=8, 1H, H-3), 8.85 (d, J_(4,5)=5.7, 1H, H-5), 9.04 (s, 1H, H-1).free base ¹³C (CDCl₃) 25.19 (C-9), 31.59 (C-8), 39.60 (C-6), 46.49(C-10), 60.54 (C-7), 123.77 (C-4), 135.65 (C-2), 136.89 (C-3), 148.06(C-5), 150.65 (C-1). Anal. Calc'd (2 HBr) C₁₀H₁₆N₂Br₂: C, 37.1; H, 5.0;N, 8.6. Found: C, 37.16; H, 4.82; N, 8.50, (M+H)⁺ 163.1240, m.p. HBr195.4° C.

3-((1-(2-hydroxyethyl)pyrrolidin-2-yl)methyl)pyridine (8): To a solutionof 3 (the free base, prepared above) (320 mg, 0.00198 M) in THF wasadded 0.33 mL (1.1 eq) of 2-benzyloxy acetaldehyde and the reactionmixture was allowed to stir at room temperature for approximately 30min. in order to allow imine formation. To this mixture was added 0.63grams (1.5 eq) of sodium triacetoxyborohydride and the reaction mixturewas allowed to continue at room temperature and monitored by TLC forcompletion. After stirring overnight the reaction was judged complete,water was added and reaction was made acidic by addition of dilutesulfuric acid (10%) and concentrated to remove the THF. The residue wasdiluted with 50 mL of water and extracted with ethyl acetate to removeany un-reacted aldehyde. The aqueous layer was made strongly basic bythe addition of 60% sodium hydroxide solution (10 mL) and extracted withethyl acetate (2×). The ethyl acetate layers were combined and washedwith brine, dried over anhydrous sodium sulfate, filtered andconcentrated to give the crude protected 4, which was used with outfurther purification. Crude protected 4 was dissolved in ethanol andsubjected to catalytic hydrogenation over 5% Pd/C and 55 psi hydrogenpressure using a Parr shaker hydrogenation apparatus. After reactioncompletion, it was diluted with chloroform, filtered though Celite toremove the catalysis and concentrated to give 8. Other compounds may besynthesized by analogy following the general chemistry pathways asdescribed above with reference to the specific chemistry described inthe above examples.

Synthesis of Pyridylpiperidine Analogs FIG. 3, Scheme 3

N-(2-Hydroxyethyl)-N′-(3-pyridylmethyl)-piperazine HCl. A solution ofN-2hydroxyethylpiperazine (3 g, 0.023 M) and 3-Pyridinecarboxaldehyde (3g 0.028 M J.2 eq.) in 200 mL of THF (dried over 4 molecular sieves) wascooled with stirring in an Ice/Salt bath to −10. To the cooled solutionwas added sodium triacetoxyborohydride (3 g, 1.5 eq.) and the solutionwas allowed to stir and warm to room temperature over night. That thistime the reaction was judged nearly complete by Thin LayerChromatography. An additional 400 mg of sodium triacetoxyborohydride wasadded and stirring continued for 2 h. The reaction was quenched by theaddition of 50 ml of 1% aqueous sulfuric acid. The reaction wasconcentrated to remove the THF, diluted with water and extracted withethyl acetate. The aqueous layer was made strongly basic (pH=10) with60% sodium hydroxide solution and extracted three times (200 ml) withethyl acetate. The ethyl acetate layers were combined, washed withbrine, dried over anhydrous sodium sulfate, filtered and concentratedunder reduced pressure to give the crude product as an oil. The oil wasdissolved in absolute ethanol and concentrated hydrochloric acid wasadded until the pH was approximately 4. The ethanol was removed underreduced pressure, which caused the HCl salt to crystallize. The solidwas recrystallized from hot isopropanol to give 5 g (66%) as the tri-HClsalt. Mp 238-239 C. Anal. Calcd for C₁₂H₁₉N₃O 3HCl: C, 43.57; H, 6.65;N, 12.71. Found: C, 43.40; H, 6.63; N, 12.62. ¹H NMR: (DMSO-d₆) (400MHz) 8: 3.31-3.84 (complex multiplet, 12H), 4.67 (s, 2H), 8.12 (m, 1H),8.86 (d, J=7.84 Hz 1H), 9.00 (d, J=5.44 Hz, 1H), 9.12 (s, H).

N-(2-Hydroxyethyl)-N′-(2-pyridylmethyl)-piperazine HCl. A solution ofN-2 hydroxyethylpiperazine (3 g, 0.023 M) and 2-Pyridinecarboxaldehyde(3 g 0.028 M J.2 eq.) in 250 mL of THF (dried over 4A molecular sieves)was cooled with stirring in an Ice/Salt bath to −10′. To the cooledsolution was added sodium triacetoxyborohydride (3 g, 1.5 eq.) and thesolution was allowed to stir and warm to room temperature over night.That this time the reaction was judged nearly complete by Thin LayerChromatography. An additional 400 mg of sodium triacetoxyborohydride wasadded and stirring continued for 2 h. The reaction was quenched by theaddition of 50 ml of 1% aqueous sulfuric acid. The reaction wasconcentrated to remove the THF, diluted with water and extracted withethyl acetate. The aqueous layer was made strongly basic (pH 10) with60% sodium hydroxide solution and extracted three times (200 ml) withethyl acetate. The ethyl acetate layers were combined, washed withbrine, dried over anhydrous sodium sulfate, filtered and concentratedunder reduced pressure to give the crude product as an oil. The oil wasdissolved in absolute ethanol and concentrated hydrochloric acid wasadded until the pH was approximately 4. The ethanol was removed underreduced pressure, which caused the HCl salt to crystallize. The solidwas recrystallized from hot isopropanol to give 4.5 g (60%) as thetri-HCl salt. Mp 210-211 C. Anal. Calcd for C₁₂H₁₉N₃O 3HCl H₂O: C,41.31; H, 6.88; N, 12.05. Found: C, 41.58; H, 6.78; N, 12.12. ′HNMR:(DMSO-d₆) (400 MHz) 8: 3.28-3.80 (complex multiplet, 12H), 4.63 (s, 2H),7.79 (m, 1H), 8.0 (d, J=7.79 Hz 1H), 8.31 (t, J=7.72 Hz, 1H), 8.83 (d,J=5.13 Hz, 1H).

N-(methyl)-N′-(3-pyridylmethyl)-piperazine HCl. A solution ofN-methylpiperazine (1 g, 0.01 M) and 3-Pyridinecarboxaldehyde (1.3 g,0.012 M, 1.2 eq.) in THF (dried over 4A molecular sieves) was cooledwith stirring in an Ice/Salt bath to −10° C. To the cooled solution wasadded sodium triacetoxyborohydride (1.4 g, 1.5 eq.) and the solution wasallowed to stir and warm to room temperature over night. That this timethe reaction was judged complete by Thin Layer Chromatography. Thereaction was quenched by the addition of 50 ml of 1% aqueous sulfuricacid. The reaction was concentrated to remove the THF, diluted withwater and extracted with ethyl acetate. The aqueous layer was madestrongly basic (pH=10) with 60% sodium hydroxide solution and extractedthree times (100 ml) with ethyl acetate. The ethyl acetate layers werecombined, washed with brine, dried over anhydrous sodium sulfate,filtered and concentrated under reduced pressure to give the crudeproduct as an oil. The oil was dissolved in absolute ethanol andconcentrated hydrochloric acid was added until the pH was approximately4. The ethanol was removed under reduced pressure, which caused the HClsalt to crystallize. The solid was recrystallized from hot ethanol togive 900 mg (30%) as the tri-HCl salt. Mp 242-243 C. Anal. Calcd forC₁₁H₁₇N₃O 3HCl 1.5H₂O: C, 40.31; H, 7.02; N, 12.82. Found: C, 40.36; H,7.01; N, 12.82. ¹H NMR: (DMSO-d₆) (400 MHz) 8: 2.85 (s, 3H) 3.60 (broadmultiplet, 8H), 4.61 (s, 2H), 8.0 (m, 1H), 8.81 (d, J=7.76 Hz I H), 8.98(d, J=5.43 Hz, 1H), 9.18 (s, 1H).

Synthesis of Scheme 4 Compounds 2-(N-methyl,N-methylphenylamino)ethan-1-ol (oxalate salt) (25)

One equivalent each of N-methylethanolamine (4.28 mL) and benzaldehyde(21, 5.42 mL) were added to a 250 mL round-bottom flask containing 25 mLof 1,2-dichloroethane (DCE). The reactants were stirred at roomtemperature for 1 hour and then 1.5 equivalents (16.94 g) of NaBH(OAc)₃were added. Stirring was continued until TLC showed the absence of allstarting materials, about 12 hours. The reaction was quenched by addingaqueous bicarbonate and stirred for 15 minutes. The solvent was removedunder reduced pressure and 10% H₂SO₄ was added to the remaining oil.This was extracted with ethyl acetate and the organic layer wasdiscarded. The pH of the water layer was changed to a slightly basic pHwith K₂CO₃ and extracted again with ethyl acetate. The organic layer wasdried over sodium sulfate and concentrated to a small volume. Theresulting free base was converted to an oxalate salt (25) andrecrystallized from isopropanol, 6.162 g, 45.29%.

m.p. 109-110° C. Anal. Calcd. for C₁₂H₁₇O₅N: C, 56.46; H, 6.71; N, 5.49.Found: C, 56.32; H, 6.66; N, 5.56. ¹H NMR: δ 7.61 (m, 2H), 7.52 (m, 3H),4.95 (bs, 1H), 4.34 (s, 2H), 3.83 (t, J=5.36 Hz, 2H), 3.21 (t, J=5.37Hz, 2H), 2.75 (s, 3H). ¹³C NMR: δ 165.14, 131.35, 129.53, 129.09, 59.43,56.75, 55.94, 40.07.

2-(N-methyl, N-methyl-4′-fluorophenylamino)ethan-1-ol (oxalate salt)(26)

A mixture of N-methylethanolamine (1.07 mL) and 22(1.43 mL) were stirredtogether in DCE for 1 hour, then 4.23 g of NaBH(OAc)₃ was added andstirring continued for 12 hours. Workup proceeded as described above.Yield: 2.48 g of 26 as the oxalate salt, 68.1%.

m.p. 140-143° C. Anal. Calcd. for C₁₂H₁₆O₅NF: C, 52.74; H, 5.90; N,5.13. Found: C, 52.78; H, 5.92; N, 5.19. ¹H NMR: δ 7.58 (m, 2H), 7.28(m, 2H), 4.91 (bs, 1H), 4.24 (s, 2H), 3.73 (t, J=5.36 Hz, 2H), 3.02 (t,J=5.37 Hz, 2H), 2.65 (s, 3H). ¹³C NMR: δ 165.07, 162.84 (J_(C-F)=245.97Hz), 133.71, 133.63, 127.83, 116.05, 115.84, 58.54, 56.72, 55.99, 40.41.

2-(N-methyl, N-methyl-3′-fluorophenylamino)ethan-1-ol (HBr salt) (27)

A mixture of N-methylethanolamine (2.14 mL) and 23 (2.82 mL) werestirred together in DCE for 1 hour, then 8.46 g of NaBH(OAc)₃ was addedand stirring was continued for 12 hours. Workup proceeded as describedfor compound 27. Yield: 5.695 g of 4.12 as the HBr salt, 89.3%.

m.p. 116-119° C. Anal. Calcd. for C₁₀H₁₅ONFBr: C, 45.45; H, 5.68; N,5.30. Found: C, 45.40; H, 5.70; N, 5.29. ¹HNMR: δ 7.51 (m, 4H), 5.45(bs, 1H), 4.48 (s, 2H), 3.89 (t, J=5.26 Hz, 2H), 3.24 (t, J=5.24 Hz,2H), 2.88 (s, 3H). ¹³C NMR: δ 162.29 (J_(c-F)=244.24 Hz), 136.17, 131.27(J_(C-F)=8.44 Hz), 127.86, 118.37 (J_(c-F)=22.21 Hz), 116.86(J_(C-F)=20.82 Hz), 58.44, 56.62, 55.37, 40.12.

2(N-methyl, N-methyl-3-pyridoamino)ethan-1-ol (oxalate Salt) (28)

A mixture of N-methylethanolamine (1.07 mL) and 24 (1.26 mL) werestirred together in DCE for 1 hour, then 4.23 g of NaBH(OAc)₃ was addedand stirring was continued for 12 hours. Workup proceeded as previouslydescribed, except that the pH flip was unnecessary. Yield: 0.399 g of 28as an oxalate salt, 11.71%.

m.p. 119-124° C. Anal. Calcd. for C₁₃H₁₈O₉N₂: C, 45.11; H, 5.29; N,8.10. Found: C, 44.93; H, 5.27; N, 8.01. ¹H NMR: δ 8.83 (m, 2H), 8.14(d, J=7.79 Hz, 1H), 7.66 (m, 1H), 5.41 (bs, 1H), 4.54 (s, 2H), 3.91 (t,J=5.05 Hz, 2H), 3.28 (t, J=5.04 Hz, 2H), 2.89 (s, 3H). ¹³C NMR: δ162.82, 152.26, 150.78, 139.51, 126.49, 124.17, 56.67, 56.59, 55.49,39.81.

2-(N-methyl, N-methylphenylamino)ethyl Acetate (Oxalate Salt) (29)

One equivalent of 25 (1.0 g), 3 equivalents of sodium acetate (0.96 g)and 20 mL of dichloroethylene were added to a 250 mL round-bottom flaskalong with 25 mL of DCE. The reaction was stirred for 30 minutes at roomtemperature, at which time 1.5 equivalents (0.6 mL) of acetic anhydridewere added. The mixture was stirred at room temperature until TLCindicated completion of reaction (about 12 hours). The reaction wasquenched with ice, then extracted with ethyl acetate. The organic layerwas washed twice with both aqueous bicarbonate and distilled water, thendried over sodium sulfate and concentrated. The resulting free base wasconverted into an oxalate salt and recrystallized from isopropanol, togive 0.84 g, (71.9%) of 29.

m.p. 109-113° C. Anal. Calcd. for C₁₄H₁₉O₆N: C, 56.56; H, 6.44; N, 4.71.Found: C, 56.57; H, 6.42; N, 4.79. ¹H NMR: δ 7.54 (m, 5H), 5.00 (bs,1H), 4.39 (t, J=5.35 Hz, 2H), 4.20 (s, 2H), 3.21 (t, J=5.34 Hz, 2H),2.65 (s, 3H), 2.13 (s, 3H). ¹³C NMR: δ 168.95, 162.77, 131.50, 129.24,127.49, 58.30, 58.01, 52.25, 38.43, 19.44.

2-(N-methyl, N-methyl-4′-fluorophenylamino)ethyl Acetate (Oxalate Salt)(30)

Above procedure was followed with 1.0 g of 26, 0.9 g of NaOAc, and 0.5mL acetic anhydride. 1.03 g of 30 was isolated as an oxalate salt,88.93%.

m.p. 135-138° C. Anal. Calcd. for C₁₄H₁₈O₆NF: C, 53.33; H, 5.75; N,4.44. Found: C, 53.30; H, 5.72; N, 4.52. ¹H NMR: δ 7.51 (m, 2H), 7.25(m, 2H), 5.49 (bs, 1H), 4.27 (t, J=5.34 Hz, 2H), 4.06 (s, 2H), 3.07 (t,J=5.32 Hz, 2H), 2.52 (s, 3H), 2.03 (s, 31-1). ¹³C NMR: δ 170.51, 164.10,162.52 (J_(C-F)=245.01 Hz), 132.93, 132.85, 126.52, 115.88, 115.67,59.71, 59.05, 53.84, 40.44, 21.01.

2-(N-methyl, N-methyl-3′-fluorophenylamino)ethyl Acetate (Maleate Salt)(31)

The procedure described in 27 was repeated with 2.58 g of 4.12, 2.21 gof NaOAc, and 1.27 mL of acetic anhydride. 1.652 g of 31 was isolated asa maleate salt, 69.0%.

m.p. 114-116° C. Anal. Calcd. for C₁₆H₂₀O₆NF: C, 56.30; H, 5.91; N,4.10. Found: C, 56.13; H, 5.82; N, 4.18. ¹H NMR: δ 7.25 (m, 1H), 7.08(m, 2H), 6.93 (m, 1H), 4.18 (t, J=5.81 Hz, 2H), 3.54 (s, 2H), 2.64 (t,J=5.81 Hz, 2H), 2.28 (s, 3H), 2.05 (3, 3H). ³¹C NMR: δ 171.38, 163.31(J_(C-F)=245.24 Hz), 141.82, 130.02 (J_(C-F)=8.17 Hz), 124.73(J_(C-F)=1.98 Hz), 115.96 (J_(c-F)=21.33 Hz), 114.33 (J_(C-F)=21.17 Hz),62.37, 62.13, 55.59, 42.84, 21.29.

Biological Data General Methods

Cell Culture: PC12 cells were maintained in 150-cm² tissue-cultureflasks in Dulbecco's Modified Eagles medium containing 7% horse serum,7% fetal calf serum, 1% non-essential amino-acids and 1% streptomycin(DMEM). The cells were incubated at 37 C in a 5% CO₂-enriched,humidified atmosphere. To attain maximum differentiation, the cells weremaintained in DMEM.NGF medium for 7 days, with the medium being changedevery 2 or 3 days.

Assay for Cell viability. Cells were dissociated by trituration andplated at 1×10⁴ cells per well on poly-L-lysine coated 96 well platescontaining DMEM.NGF media. Next, the difrerentiated cells were incubatedwith the test drug at different concentrations for the specified periodof time. Cell viability (cytotoxicity) was determined using the CellTiter 96 non-radioactive cell proliferation/cytotoxicity assay kit(Promega), which is based on the cellular conversion of a3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) intoa formazan product which can be detected spectrophotometrically. Afterdeprivation of cells from NGF and serum, the medium was aspirated and 15l of dye solution dissolved in DMEM was added. After an additional 4 hrincubation at 37 C, 100 l of solubilization/stop solution was added andthe absorbance of solubilized MIT formazan products was measured at 570nm.

Binding assay: PC-12 cells were plated at 100,000 cells per well onpoly-L-lysine coated 24 well plates containing DMEM.NGF solution. Toattain maximum differentiation, culture medium was replaced every 48-72hours with fresh DMEM.NGF solution, and the cells were maintained for 7days. Saturation binding studies were performed with [¹²⁵I]-bungarotoxinconcentration ranging from 0.1 nM to 10 nM to obtain ligand Kd.Competition binding assays were performed with 10-15 concentrations ofeach analog. For the binding studies, the culture medium was removed andreplaced with medium containing an analog, and 2 nM [¹²⁵I]-bungarotoxin.The non-specific binding was determined in the presence of 1 Munlabeled-bungarotoxin, which was added prior to the[¹²⁵I]-bungarotoxin. The cells were incubated for 2 hr at 37 C andrinsed four times with 2 ml aliquots of HEPES solution (137 mM NaCl, 5.4mM Kcl, 0.8 mM MgSO₄, 0.9 mM NaPO₄, 0.4 mM K₂PO₄, 1.8 mM CaCl₂, 2 mg/mlBSA, and 10 mM HEPES, pH 7.4). After washing, cells were detached fromthe plate by addition of 1 ml of 1N NaOH. Bound radioactivity wasquantified by gamma counting. Nonspecific binding, determined in thepresence of excess unlabelled-bungarotoxin, was subtracted from totalbinding to yield specific binding. Results, were normalized to totalprotein, which was assayed as follows: the cells were scraped in 2%SDS/0.1N NaOH solution and the BCA protein assay was conducted with BSAas a standard. K_(i) values were calculated using the Cheng and Prusoffequation: K_(i)=IC50/(1+[L}/K_(d)) (where [L] is th concentration ofradioligand. See Cheng and Prusoff, Biochem. Pharmacol. 23, 3099-3108,1973.

Findings

Differentiated PC-12 cells were deprived of neurotrophic factors for 24hr to promote cytotoxicity. Cell viability was measured by the MIT assayunder control conditions, and in cultures in which a choline analog wasintroduced into the medium at the time of neurotrophic factorwithdrawal. The data for choline and 6 other analogs are presented inFIG. 5. The analog benzylcholine did not affect cell viability. Cholineand its mono-, di-, and tri-ethyl analogs produced a concentrationdependent and intermediate level of neuroprotection improving cellviability by up to 40% of baseline. The analogs pyrrolidinecholine andacetylpyrrolidinecholine were most effective, improving cell viabiltiyby over 60% of baseline. In fact, these two analogs were more potent aswell, since the effects produced by the lowest dose tested (0.1 mM) werenot that much reduced from the effects attributed to the 10 mMconcentration.

Since choline may serve as a natural ligand for the 7 subtype of thenicotinic acetyl choline receptor, and since stimulation of the 7subtype by nicotine is known to produce a neuroprotective effect, wenext sought to determine whether the neuroprotective effects produced bycholine could be blocked by nicotinic receptor antagonists. The data arepresented in FIG. 6. Co-administration of choline and the non-selectiveantagonist mecamylamine resulted in a significant (and almost complete)inhibition of choline's neuroprotective action. A similar finding wasobtained for those experiments in which the 7 subtype preferringantagonist, MLA, was used.

Since the neuroprotective action to choline appeared to be mediatedthrough stimulation of cell surface 7 nicotinic actylcholine receptors,we measured the ability of choline to displace the cell surface bindingof [¹²⁵I]-bungarotoxin (a selective 7 nicotinic acetylcholine receptorantagonist) to differentiated PC-12 cells. These data are presented inFIG. 7 along with the displacement curve for nicotine as a comparison.Choline was only 50 fold less potent than nicotine in displacing[125I]-bungarotoxin.

Next, pyrrolidinecholine, the most active choline analog, was comparedwith benzylcholine, the inactive choline analog in the neuroprotectionassay, for their ability to displace the cell surface binding of[¹²⁵I]-bungarotoxin to PC-12 cells. As indicated in FIG. 8, the inactiveanalog (for neuroprotection), benzylcholine also failed to significantlyinteract with [¹²⁵I]-bungarotoxin binding sites. In contrast,pyrrolidinecholine fully displaced [¹²⁵I]-bungarotoxin binding, and itexhibited a slightly more potent affinity for the site than did cholinewhich fits with pyrrolidinecholine's greater activity in theneuroprotrection assay. As indicated in FIGS. 9 and 10, theneuroprotective action of pyrrolidinecholine (like choline) (FIG. 9) wasblocked by pretreatment with either mecamylamine (10 M) or MLA (10 nM)(FIG. 10).

Further Experiments Cytoprotective Effect of Choline and CompoundsAccording to the Present Invention

Cytoprotective effect of nicotine in differentiated PC-12 cells.Differentiated PC-12 cells described above were exposed to the indicatedconcentrations of nicotine 24 hours (see FIG. 11) prior to growth factorwithdrawal (removal of nerve growth factor and changing fromserum-containing to serum-free medium. % Protection values werecalculated as the ratio of ELISA-based absorbance values for [protectedcells-deprived cells (no nicotine): control (non-deprived)cells−deprived cells]×100. Each point represents data obtained from twoseparate assays each performed with 5-8 replicates per point. Note thebiphasic nature of the concentration-effect curve in FIG. 11.

Cytoprotective Effect of Choline Analogs in Differentiated PC-12 Cells.

The above-described differentiated PC-12 cells were exposed to theindicated concentrations (see FIGS. 13A and B) of the FIG. 12 cholineanalogs 24 hours prior to growth factor withdrawal (removal of nervegrowth factor, and changing from serum-containing to serum-free medium).% Protection values were calculated as the ratio of ELISA-basedabsorbance values for [protected cells−deprived cells (no analog):control (non-deprived) cells−deprived cells]×100. Each data point inFIG. 13 represents data obtained from two separate assays each performedwith 5-8 replicates per point. Note that Compound 27, and Compound 2provide results similar to nicotine (FIG. 11).

It is to be understood by those skilled in the art that the foregoingdescription and examples are illustrative of practicing the presentinvention, but are in no way limiting. Variations of the detailpresented herein may be made without departing from the spirit and scopeof the present invention as defined by the following claims.

1. A compound according to the chemical structure:

Where each of R¹, R² and R⁴ is independently selected from H, a C₁ toC₁₂ straight, branch-chained or cyclic saturated or unsaturatedhydrocarbon, a (CH₂)_(n)OR⁵ group or an R⁶ group; R³ is H, a C₁ to C₁₂alkyl or alkene group or a (CH₂)_(n)OR⁵ group with the proviso that R³and R⁴ are not both H; R^(A) and R^(B) are independently selected fromH, a C₁ to C₁₂ alkyl or alkenyl group or a (CH₂)_(m)OR⁵ group,preferably with the proviso that when one of R^(A) or R^(B) is a(CH₂)_(m)OR⁵ group, the other of R^(A) or R^(B) is not a (CH₂)_(m)OR⁵group; R⁵ is H or a C₁ to C₁₂ alkyl, alkenyl group or carboxylic acid, aC₂ to C₁₂ acyl or alkyl ester group or a C₃ to C₁₂ alkylene ester group;R⁶ is a group according to the structure:

Z is

n is 0 to 12; m is 1 to 8; and X₁, X₂ and X₃ are each independentlyselected from H, OH, F, Cl, Br, R⁴, OR⁴, CF₃ or OCF₃; or apharmaceutically acceptable salt thereof.
 2. The compound according toclaim 1 wherein n is 1 to
 8. 3. The compound according to claim 1wherein said compound has the structure:

R^(A) and R^(B) are independently selected from H, a C₁ to C₃ alkylgroup or a (CH₂)_(m)OR⁵ group, with the proviso that when one of R^(A)or R^(B) is a (CH₂)_(m)OR⁵ group, the other of R^(A) or R^(B) is not a(CH₂)_(m)OR⁵ group; R⁵ is H or a C₁ to C₄ alkyl or alkylene ester group;R⁶ is a group according to the structure:

Z is □(CH₂)_(n)—; X₁, X₂ and X₃ are selected from H, F, CL, Br, NO₂ orCF₃; n is 1-4 and m is 1-4 or a pharmaceutically acceptable saltthereof.
 4. The compound according to claim 3 where one of X₁, X₂ and X₃is F and the other of X₁, X₂ and X₃ is H.
 5. The compound according toclaim 4 wherein R⁶ is


6. The compound according to claim 5 wherein m is 1-2, n is 1-2, R^(A)is CH₃ and R^(B) is CH₂CH₂OH.
 7. The compound according to claim 6wherein m is 1 and n is
 2. 8. The compound according to claim 3 whereinR⁶ is

and n is 1-2, m is 1-2.
 9. The compound according to claim 8 whereinR^(A) is CH₃ and R^(B) is CH₂CH₂OH.
 10. The compound according to claim3 wherein R⁶ is

X₁, X₂ and X₃ are selected from H, F, Cl, Br, NO₂ or CF₃; m is 1-2, n is1-2, R^(A) is CH₃ and R^(B) is CH₂CH₂OR.
 11. The compound according toclaim 10 wherein R is a C₃-C₆ alkylene ester group.
 12. The compoundaccording to claim 11 wherein X₁, X₂ and X₃ are H.
 13. The compoundaccording to claim 1 wherein said chemical structure is

where R′ and R² are H; R³ is a (CH₂)_(n)OR⁵ group; and R⁴ is H or agroup according to the structure:

Z is □(CH₂)_(n)— and n is 1-2.
 14. The compound according to claim 13wherein R⁵ is H.
 15. A pharmaceutical composition comprising aneffective amount of a compound according to the chemical structure:

Where each of R′, R² and R⁴ is independently selected from H, a C₁ toC₁₂ straight, branch-chained or cyclic saturated or unsaturatedhydrocarbon, a (CH₂)_(n)OR⁵ group or an R⁶ group; R³ is H, a C₁ to C₁₂alkyl or alkene group or a (CH₂)_(nOR) ⁵ group with the proviso that R³and R⁴ are not both H; R^(A) and R^(B) are independently selected fromH, a C₁ to C₁₂ alkyl or alkenyl group or a (CH₂)_(m)OR⁵ group,preferably with the proviso that when one of R^(A) or R^(B) is a(CH₂)_(m)OR⁵ group, the other of R^(A) or R^(B) is not a (CH₂)_(m)OR⁵group; R⁵ is H or a C₁ to C₁₂ alkyl, alkenyl group or carboxylic acid, aC₂ to C₁₂ acyl or alkyl ester group or a C₃ to C₁₂ alkylene ester group;R⁶ is a group according to the structure:

Z is

n is 0 to 12; m is 1 to 8; and X₁, X₂ and X₃ are each independentlyselected from H, OH, F, Cl, Br, R⁴, OR⁴, CF₃ or OCF₃; or apharmaceutically acceptable salt thereof, optionally in combination witha pharmaceutically acceptable additive, carrier or excipient.
 16. Thecomposition according to claim 15 wherein n is 1 to
 8. 17. Thecomposition according to claim 15 wherein said compound has thestructure:

R^(A) and R^(B) are independently selected from H, a C₁ to C₃ alkylgroup or a (CH₂)_(m)OR⁵ group, with the proviso that when one of R^(A)or R^(B) is a (CH₂)_(m)OR⁵ group, the other of R^(A) or R^(B) is not a(CH₂)_(m)OR⁵ group; R⁵ is H or a C₁ to C₄ alkyl or alkylene ester group;R⁶ is a group according to the structure:

Z is □(CH₂)_(n)—; X₁, X₂ and X₃ are selected from H, F, Cl, Br, NO₂ orCF₃; n is 1-4 and m is 1-4 or a pharmaceutically acceptable saltthereof.
 18. The composition according to claim 17 where one of X₁, X₂and X₃ is F and the other of X_(i), X₂ and X₃ is H.
 19. The compositionaccording to claim 18 wherein R⁶ is


20. The composition according to claim 19 wherein m is 1-2, n is 1-2,R^(A) is CH₃ and R^(B) is CH₂CH₂OH.
 21. The composition according toclaim 20 wherein m is 1 and n is
 2. 22. The composition according toclaim 17 wherein R⁶ is

and n is 1-2, m is 1-2.
 23. The composition according to claim 22wherein R^(A) is CH₃ and R^(B) is CH₂CH₂OH.
 24. The compositionaccording to claim 17 wherein R⁶ is

X₁, X₂ and X₃ are selected from H, F, Cl, Br, NO₂ or CF₃; m is 1-2, n is1-2, R^(A) is CH₃ and R^(B) is CH₂CH₂OR.
 25. The composition accordingto claim 24 wherein R is a C₃-C₆ alkylene ester group.
 26. Thecomposition according to claim 25 wherein X₁, X₂ and X₃ are H.
 27. Thecomposition according to claim 15 wherein said chemical structure is

where R′ and R² are H; R³ is a (CH₂)_(n)OR⁵ group; and R⁴ is H or agroup according to the structure:

Z is □(CH₂)_(n)— and n is 1-2.
 28. The composition according to claim 27wherein R⁵ is H.
 29. A method of treating a patient having aneurodegenerative condition or other neurological condition whereacetylcholine transmission neurons and their target cells are affectedcomprising administering to said patient an effective amount of acompound according to the chemical structure:

Where each of R′, R² and R⁴ is independently selected from H, a C₁ toC₁₂ straight, branch-chained or cyclic saturated or unsaturatedhydrocarbon, a (CH₂)_(n)OR⁵ group or an R⁶ group; R³ is H, a C₁ to C₁₂alkyl or alkene group or a (CH₂)_(nOR) ⁵ group with the proviso that R³and R⁴ are not both H; R^(A) and R^(B) are independently selected fromH, a C₁ to C₁₂ alkyl or alkenyl group or a (CH₂)_(m)OR⁵ group,preferably with the proviso that when one of R^(A) or R^(B) is a(CH₂)_(m)OR⁵ group, the other of R^(A) or R^(B) is not a (CH₂)_(m)OR⁵group; R⁵ is H or a C₁ to C₁₂ alkyl, alkenyl group or carboxylic acid, aC₂ to C₁₂ acyl or alkyl ester group or a C₃ to C₁₂ alkylene ester group;R⁶ is a group according to the structure:

Z is

n is 0 to 12; m is 1 to 8; and X₁, X₂ and X₃ are each independentlyselected from H, OH, F, Cl, Br, R⁴, OR⁴, CF₃ or OCF₃; or apharmaceutically acceptable salt thereof.
 30. The method according toclaim 29 wherein n is 1 to
 8. 31. The method according to claim 29wherein said compound has the structure:

R^(A) and R^(B) are independently selected from H, a C₁ to C₃ alkylgroup or a (CH₂)_(m)OR⁵ group, with the proviso that when one of R^(A)or R^(B) is a (CH₂)_(m)OR⁵ group, the other of R^(A) or R^(B) is not a(CH₂)_(m)OR⁵ group; R⁵ is H or a C₁ to C₄ alkyl or alkylene ester group;R⁶ is a group according to the structure:

Z is □(CH₂)_(n)—; X₁, X₂ and X₃ are selected from H, F, Cl, Br, NO₂ orCF₃; n is 1-4 and m is 1-4 or a pharmaceutically acceptable saltthereof.
 32. The method according to claim 31 where one of X_(i), X₂ andX₃ is F and the other of X₁, X₂ and X₃ is H.
 33. The method according toclaim 32 wherein R⁶ is


34. The method according to claim 33 wherein m is 1-2, n is 1-2, R^(A)is CH₃ and R^(B) is CH₂CH₂OH.
 35. The method according to claim 34wherein m is 1 and n is
 2. 36. The method according to claim 31 whereinR⁶ is

and n is 1-2, m is 1-2.
 37. The method according to claim 36 whereinR^(A) is CH₃ and R^(B) is CH₂CH₂OH.
 38. The method according to claim 31wherein R⁶ is

X₁, X₂ and X₃ are selected from H, F, Cl, Br, NO₂ or CF₃; m is 1-2, n is1-2, R^(A) is CH₃ and R^(B) is CH₂CH₂OR.
 39. The method according toclaim 38 wherein R is a C₃-C₆ alkylene ester group.
 40. The methodaccording to claim 39 wherein X₁, X₂ and X₃ are H.
 41. The methodaccording to claim 28 wherein said chemical structure is

where R¹ and R² are H; R³ is a (CH₂)_(n)OR⁵ group; and R⁴ is H or agroup according to the structure:

Z is □(CH₂)_(n)— and n is 1-2.
 42. The method according to claim 41wherein R⁵ is H.
 43. The method according to claim 29 wherein saidcondition is selected from the group consisting of Parkinson's disease,Huntington disease, Alzheimer's disease, amyotrophic lateral sclerosis,spinal muscular atrophy, Friedrich's ataxia, Pick's disease,Bassen-Kornzweig syndrome, Refsom's disease, retinal degeneration,Cruetzfelt-Jacob syndrome or prion disease, dementia with Lewy bodies,schizophrenia, paraneoplastic cerebellar degeneration andneurodegenerative conditions caused by stroke.
 44. The method accordingto claim 31 wherein said condition is selected from the group consistingof Parkinson's disease, Huntington disease, Alzheimer's disease,amyotrophic lateral sclerosis, spinal muscular atrophy, Friedrich'sataxia, Pick's disease, Bassen-Kornzweig syndrome, Refsom's disease,retinal degeneration, Cruetzfelt-Jacob syndrome or prion disease,dementia with Lewy bodies, schizophrenia, paraneoplastic cerebellardegeneration and neurodegenerative conditions caused by stroke.
 45. Themethod according to claim 34 wherein said condition is selected from thegroup consisting of Parkinson's disease, Huntington disease, Alzheimer'sdisease, amyotrophic lateral sclerosis, spinal muscular atrophy,Friedrich's ataxia, Pick's disease, Bassen-Kornzweig syndrome, Refsom'sdisease, retinal degeneration, Cruetzfelt-Jacob syndrome or priondisease, dementia with Lewy bodies, schizophrenia, paraneoplasticcerebellar degeneration and neurodegenerative conditions caused bystroke.
 46. The method according to claim 38 wherein said condition isselected from the group consisting of Parkinson's disease, Huntingtondisease, Alzheimer's disease, amyotrophic lateral sclerosis, spinalmuscular atrophy, Friedrich's ataxia, Pick's disease, Bassen-Kornzweigsyndrome, Refsom's disease, retinal degeneration, Cruetzfelt-Jacobsyndrome or prion disease, dementia with Lewy bodies, schizophrenia,paraneoplastic cerebellar degeneration and neurodegenerative conditionscaused by stroke.
 47. The method according to claim 39 wherein saidcondition is selected from the group consisting of Parkinson's disease,Huntington disease, Alzheimer's disease, amyotrophic lateral sclerosis,spinal muscular atrophy, Friedrich's ataxia, Pick's disease,Bassen-Kornzweig syndrome, Refsom's disease, retinal degeneration,Cruetzfelt-Jacob syndrome or prion disease, dementia with Lewy bodies,schizophrenia, paraneoplastic cerebellar degeneration andneurodegenerative conditions caused by stroke.